Substituted pteridines



United States Patent SUESTETUTED PTERIDINES Meyer Sietzinger, NorthPlainfield, and Max Tishler,

Westfield, N. l, assignors to Merck & Co., Inc,, Rahway, N. 1., acorporation of New Jersey No Drawing. Application May 3, 1954, SerialNo. 427,339

6 Claims. (Cl. 260251.5)

This invention relates to pteroylglutamic acid, commonly known as folicacid. More particularly, it is concerned with synthetic methods ofproducing pteroylglutamic acid and novel intermediates useful therein.

Pteroylglutamic acid, or vitamin B occurs naturally in yeast, liver,grasses, and mushrooms. This substance has been found to betherapeutically efiective in the treatment of macrocytic anemias, sprue,and other conditions of the circulatory system. Although pteroylglutamicacid can be isolated from natural sources, it has been found thatchemical synthesis of the vitamin is a more desirable method ofproduction. However, the syntheses of pteroylglutamic acid reportedheretofore have not been entirely satisfactory due to low yields andinherent diificulties caused by the very low solubility of theintermediates employed.

It is therefore an object of this invention to provide a new and novelsynthesis of pteroylglutamic acid. Another object is to provide aprocess for producing pteroylglutamic acid from readily available,inexpensive startingmaterials. A further object is to provide a processfor producing pteroylglutamic acid wherein crystalline, solubleintermediates are employed. It is also an object to provide new andnovel intermediates useful in preparing pteroylglutamic acid. Otherobjects will be apparent from the following detailed description of theinvention.

According to a prefen-ed embodiment of the present invention, it hasbeen discovered that these objects and others can be achieved whenpteroylglutamic acid is produced by the process which comprises reactinga 1,2-.di halo-1,3,3-trisubstituted propane wherein the 1,3,3substituents are alkoxy, aryloxy or aralkoxy radicals (I) with a weakbase to produce the corresponding 2-halo- 3,3-dialkoxy, diaryloxy, ordiaralkoxy propionaldehyde (II), condensing the resultingpropionaldehyde with a 2,4,5-triamino pyrimidine substituted in the 6position with a hydroxy, alkoxy, aryloxy, or aral koxy radical (III) toproduce a Z-amino-S,6-dihydropteridine of the formula wherein R is analkoxy, aryloxy, or aralkoxy radical and R is a hydroxy, alkoxy, aryloxyor aralkoxy radical (IV), oxidizing the substituted2-amino-5,6-dihydropteridine to the corresponding Z-amino pteridine (V),acylating the said substituted Z-arnino pteridine to produce thecorresponding Z-acylamino pteridine (V1,) and subseguently hydrolyzingit to produce a 2 acylamin o-4-hydroxy-6-formylpteridine (VII), reactingthe 2-acylarnino 4 hydroXy-6-forn1ylpteridine with L-(-1)N-(p-amiuobenzoyl) glutamic acid to produce the Schifis base N-{p [(2acylarnino 4-hydroxy-6-pteridylrnethylene) imino] benzoyl} glutamic acid(IX), reducing said Schiifs base to N -acyl-N -formyl pteroylglutamicacid (X), and hydrolyzing said compound to pteroylglutamic acid (XI).This sequence of reactions can be illustrated as follows:

NHQ

n,N- Nn,

(ancncnonn (R)2CHCHCHO (III) x X X (I) (11 a n R I A r H I N k flum 0mm.5 i 7) l HEN \gl, H2N \N (IV) v r e N N m Team Tenn I l RzHN- N mnN\N/*\Ny (VII) 1 coon (VI +H2NCONHCH t on, CH1

coon

(VIII) S I w /N\ on=N -ooNn ii i on,

on, R aHN N N coon 1 one /N coon N CHzN -CONHGH on, RHYK on;

a 1 y coon en N coon l CHaNH-Q-CONHCH CH2 HN-k bn P teroylglutamic acidwherein X is a halogen, R is an alkoxy, aryloxy, or aralkoxy radical, Ris a hydroxy, alkoxy, aryloxy, or aralkoXy radical, and R is an acylradical.

The starting materials which can be used in practicing this inventionare the 1,2-dihalo-1,3,3-trialkoxy propanes, l,Z-dihalo-1,3,3-triaryloxypropanes, and the 1,2-dihalo- 1,3,3-triaralkoxy propanes. Such compoundsare readily prepa edby halogenating a 1,3,3-trialkoxy, triaryloxy, or

in the I. Am. Chem. Soc., 73, 206 (1951). For exam le,l,2-dibromo-l,3,3-triethoxy propane is produced by reacting1,3,3-triethoxy propylene-l with bromine in an inert solvent. In asimilar manner, l,2-dibronto-l;,3-,3-tribenzyloxy propane is prepared byreacting 'acrolein dibromide with benzyl alcohol in the presence ofHydrochloric acid to form 2-bromo-1,3,3*tribenz yloxy propane, reactingsaid compound with an inorganic base. in the presence of benzyl alcoholto produce 1,3,3-tribenzyloxy propylene-l which is then r'eadilybrominated to the desired 1,2- -dibromo-1,3,3-tribenzyloxy propane.Other compounds which can be prepared according to this method by use'of the corresponding alcohol and halogen are1,2-dichloro-1,3,3-trimethoxy propane, 1,2-dichloro-1,3,3-tribu toxypropane, and 1,2-dibromo-1,3,3-tripropoxy propane. After the reactionhas been terminated the 1,2-dihalo-1,3,3-trisubstituted propane can beisolated from the reaction mixture and purified or the reaction mixturecan be used directly as the starting material in our process withoutfurther processing.

In the first step of the process for preparing pteroylglutamic acidaccording to the preferred method of this invention, a2-halo-3,3-dialkoxy, diaryloxy, or diaialkoxy propionaldehyde isproduced by reacting the corresponding 1,Z-dihalo-1,3,3-trialkoxy,triaryloxy, or triaralkoxy propane with a weakly basic substance.Examples of weak bases which are suitable for effecting the reaction aresodium acetate, sodium bicarbonate, ammonium hydroxide, and the like.The reaction is conveniently conducted in the presence of a suitableinert solvent or mixture of solvents. In general, hydroxylated solventssuch as the alcohols are not used as the reaction medium since suchsolvents are not completely inert under the conditions of reaction. Someexamples of solvents which can be used are water, dioxane, ether,chloroform, carbon tetrachloride, acetone, benzene and formamide. Thereaction proceeds satisfactorily Within a wide range of temperatures.Thus lowered temperatures of about C. and elevated temperatures such as80 C. can be used with good results. The resulting 2halo-3,3-dia1koxy,diaryloxy, 'or diar'alkoxy propionaldehyde can be recovered from thereaction mixture by conventional methods. Examples of the novelcompounds which can be prepared by this process are 2-bromo-3,3-diethoxypropionaldehyde, 2 bromo 3,3 dibenzyloxy propionaldehyde, 2- chloro-3,3iipropoxy propionaldehyde, and 2-bromo-3,3- dibutoxy propionaldehyde.

The pteridine moiety, which is an essential structural unit ofpteroylglutamic acid, is produced by condensinga 2-halo-3,3-dialkoxy,diaryloxy, or diaralkoxy propionaldehyde with a 2,4,5-triaminopyrimidine having in the 6 position a hydroxy, alkoxy, aryloxy, oraralkoxy radical to produce the corresponding 2-amino-6-dialkoxy-methyl,diaryloxymethyl, of diaralkoxymethyl-5,6-dihydro-pteridine. Fortunately,the resulting pteridine moiety is practically all the 6-methyl positionisomer needed in the synthesis of biologically active compounds such aspteroylglutamic acid. The condensation is preferably eflected byintimately contacting the reactants in the pres ence of a solvent and acondensing agent. The solvent can be water or an inert organic solventor mixture of suchsolvents. Illustrative of solvents which can be usedare hydroxylated solvents such as alcohols and glycols, particularlyethyl alcohol and ethylene glycol, and solvents such as acetone,benzene, and formamide. Examples of condensing agents which might bementioned are sodium acetate, disodium phosphate, silver carbonate, andsodium formate. The reaction will proceed at ordinary temperatures butthe rate of reaction may be increased by using elevated temperaturessuch as 60 C.

The condensation resulting in formation of the pteridine nucleusproceeds satisfactorily regardless of the substituent in the 6 positionof the 2,4,5-triamino pyrimidine used as reactant. Thus, equally goodresults are obtained when the G-substituent is a hydroxy, an alkoxyradical such as ethox y, propox-y or butoxy, aryloxy radical such asphenoxy, or an aralkoxy radical such as benzyloxy. However, thecondensation is most easily accomplished when a 2-halo-3,3-dialkoxy,diaryloxy, or diaralkoxy propionaldehyde is used in which the halogen isbromine or chlorine. In specific embodiments of this condensation2-amino-4 benzyloxy 6 diethoxymethyl 5,6 dihydropteridine and2-amino-4-hydroxy-6-diethoxymethyl 5,6- dihydropteridine can be producedby condensing respectively 2,4,5-triarnino-6-benzyloxy pyrimidine and2,4,5 triamino-6-hydroxy pyrimidine with 2-broir10-3,3-diethoxypropionaldehyde in the presence of aqueous ethanol and sodium acetate.After completion of the condensation the desired product can berecovered from the reaction mixture by conventional procedures or thereaction mixture can be used directly in the preparation of the fullyaromatic pteridine moiety.

The fully aromatic 2-amino pteridines having a droxy, alkoxy, aryloxy,or ara lkoxy radical in the 4 position and a dialkoxymethyl,diaryloxymethyl, or diaralko'x'ymethyl radical in the 6 position can beprepared by dehydrogenation of the correspondingly substituted 5,6-dihydropteridines. The dehydrogenation is readily accomplished byintimately contacting the 5,6-dil1'ydropteridinewith a mild oxidizingagent. Specific examples of suitable oxidizing agents are air, oxygen,iodine, and hydrogen peroxide with an iron catalyst. In general, ll; ispreferable to maintain a pH of about 8 to 9 to obtain best results. Inaddition, the reaction is convemently accomplished in a suitable inertsolvent such as alcohols, glycols, acetone, benzene, formamide, dioxane,and water. The resulting pteridines can be isolated from the reactionmixture by conventional methods. According to specific applications ofthis dehydrogenation reaction 2- amino-4-benzyloxy 6 diethoxymethylpteridine and 2- amino-4-hydroxy-6-diethoxyrnethyl pteridine areproduced by oxidizing respectively2-amino-4-benzyloxy-6-d1ethoxymethyl-5,6-dihydropteridine and Z-amino 4hydroxy-6 diethoxy-methyl-5,G-dihydropteridine with hydrogen peroxideand ferrous sulfate in a suitable solvent. Examples of otherrepresentative pteridines which can be prepared in this manner are2-amino-4-butoxy-6-dibenzyloxyrr ethyl pteridine,2-amino-4-ethoxy-6-dimethoxymethyl pteridine, and2-amino-4-methoxy-6-dipropoxymethyl pteridine.

In the next step of the process 2-arnino pteridines containing ahydroxy, alkoxy, aryloxy, or aralkoxy substituentin the 4 position and adialkoxymethyl, diaryloxymethyl, or diaralkoxymethyl substituent in the6 position are converted to the corresponding novel 2-acylaminopteridines. The acylation can be effected by intimately contacting thesubstituted Z-amino peteridines with a suitable acylating agent such asan acyl halide or carboxylic acid anhydride. Acethyl chloride, propionylchloride, butyryl chloride, benzoyl bromide, acetic anhydride, propionicanhydride, butyric anhydride, and benzoic anhydride are examples ofsuitable acylating agents. The acylation is conducted in a. liquidreaction medium which can be an inert organic solvent or an excess ofthe acylating agent. In general, an added solvent is not required sincethe acylating agents are usually liquids at normal or slightly elevatedtemperatures. Although the reaction proceeds at ordinary temperatures itis usually effected at higher temperatures such as the refluxtemperature to enhance the rate of reaction. The desired 2-acylaminopteridine can be isolated from the reaction mixture by conventionalmethods such as cooling and filtering to separate the crystallineproduct. According to this acylation procedure, Z-acylamino pteridineshaving the described substituents in the 4 and 6 positions can bereadily prepared wherein the acyl substituent is an alkyl, aryl, oraralkyl carbonyl radical. Thus. the specific 2-acylamino pteridineswhich can be produced according to this process are 2-propionamido-4-benzyloxy-6-dimethoxymethyl pteridine, 2-butyramido-4-hydroxy-6-dipropoxymethyl pteridine, 2-acetamido-4-phenoxy-6-diethoxyniethyl pteridine, 2-acetamido-4-hydroxy-6-diethoxymethyl' pteridine,2-benzamido-4ehydroxy-6-diethoxymethyl pteridine, andZ-phenylacetamido-4-hydroxy-6-diethoxymethyl pteridine.

The acylated pteridines possess unique and valuable properties whichdistinguish them from the non-acylated pteridines. For example, thenon-acylated pteridines reported in the art are amorphous compoundswhichare nearly insoluble in ordinary solvents. Therefore it was indeedsurprising to discover that acylated pteridines, and derivatives ofacylated pteridines, could be readily produced in crystalline form.Furthermore, the acylated pteridines were found to have an unexpectedlyhigh solnbi-lityin Water and'many organic solvents. The ability toproduce crystalline compounds With high solubilityby the introduction ofan'acyl group on the Z-amino was entirely unexpected since pteridineshaving such desirable properties were heretofore unknown. Thiscombination of desirable properties greatly enhances the usefulness ofthe Z-acylamino pteridines. Thus, the production of crystallinepteridines is a great aid in the puri fication of such compounds.Because of their greater solubility, the 2-acylamino pteridines can beused in reactions with smaller volumes of solvents than the non--acylated compounds thereby allowing a saving in material and permittinggreater manipulative freedom.

In the next step of the process, 2-.acylamino pteridines substituted inthe 4 position with a hydroxy, alkoxy, aryloxy, or aralkoxy radical andin the 6 position with a dialkoxymethyl, diaryloxymethyl, ordiaralkoxymethyl radical are hydrolyzed to the corresponding2.acylamino-4-hydroxy-6-formyl pteridine. According to this hydrolysisreaction the acetal in the 6 position is converted to a formyl radical.Simultaneously pteridines which contain an alkoxy, aryloxy, or aralkoxyradical in the 4 position are hydrolyzed to the corresponding 4 hydroxypteridines. Either mineral or organic acids can be used for thehydrolysis. Examples of some suitable acids are hydrochloric acid,sulfuric acid, phosphoric acid, acetic acid, and formic acid. Thereaction is readily conducted in a solvent medium which can be an excessof the acid used or an added solvent such as Water or an inert organicsolvent. Normal or somewhat elevatedtemperatures may be used to promotethe reaction. After the hydrolysis has been completed crystallineZ-acylaminolhydroxy-6-formyl pteridine is isolated by conventionalmethods. In specific applications of this reactionZ-acetamido-4-hydroxy-o-diethoxymethyl pteridine and2-propion'amido-4-benzyloxy-6-dimethoxymethyl pteridine are hydrolyzedwith formic acid to 2-acetamido-4-hydroxy- 6-formyl pteridine and2-propionamidol-hydroxy-6- formyl pteridine. Other similar compoundswhich can be prepared in this manner are 2-benzamido-4-hydroxy- 6-formylpteridine, Z-butyrarnido-4-hydroxy-6-formyl pteridine and2-phenyl-acetamido-4-hydroxy-6-formyl pteridine.

In the succeeding step the Schiifs baseN-{p-KZ-acylamino-4-hydroxy-6-pteridylmethylene) imino] benzoyl}glutamic acid is produced by contacting the Z-acylamino-4-hydroxy-6-formyl pteridines with L-(-1)N-(p-aminobenzoyl} glutamicacid in the presence of a solvent. In general, inert organic solventscan be used for the reaction medium. Some examples of suitable solventsare the alcohols such as methanol, ethanol, propanol and dioxane. Thereaction can be carried out at room temperature or more elevatedtemperatures. By cooling the mixture after the reaction has beencompleted the desired Schiifs base crystallizes from solution and isrecovered by conventional methods. According to this procedure N-{p-[(2acylamino 4 hydroxy 6-pteridylmethylene) imino] benzoyl} glutamic acidscan be prepared wherein the acyl group is an 'alkyl carbonyl, arylcarbonyl or aralkyl carbonyl radical by selecting the corresponding 2-aeylamino-4-hydroxy-6-formyl pteridines for the reaction.

Representative of the broad. class of Schifi s, bases can beprepared'bythis process are N {pE'('2-acetamid4-hydroxy-G-pteridylmethylene) imino] benzoyl} glutamic 'acid,N-{p-I(2-benzamido-4-hydroxy-6-pteridylmethylene) imino] benzoyl}glutamic acid and N-{p-[(2- butyramido-4 hydroxy 6 pteridylrnethylene)imino] benzoyl} glutamic acid.

According to the next-step of the preferred process N acyl-N -formylpteroylglutamic acid is produced wherein the acyl substituent is analkyl carbonyl, aryl carbonyl or aralkyl carbonyl radical by contactingthe N-{p-[(2- acylamino-4-hydroxy-6pteridylrnethylene) imino] benzoyl}glutamic acid with formic acid or aproduct which generates formic acidin situ. The reaction is conveniently conducted in the presence of asolvent. Examples of suitable solvents are the hydroxylated solventssuch as the lower alcohols, dioxane, ether, and the inert chlorinatedhydrocarbons. In addition, an excess of formic acid may be used for thereaction medium. A small amount; of acetic anhydride is ordinarily addedin effecting the reaction since it serves to promote high yields of thedesired N -acyl-N -formyl pteroylglutamic acids. Although the reactionproceeds at ordinary temperatures the rate of reaction is increased atelevated temperatures. After the reaction has been completed the desiredproduct can be isolated-by cooling the reaction mixture and adding etherto precipitate the product. In one embodiment of this method N -acetyl-N-formyl pteroylglutamic acid is produced by reactingN-{p-[(2-acetamido-4-hydroxy-6- pteridylrnethylene) imino] benzoyl}glutamic acid in dioxane with formic acid. Representative of othercornpounds which can be prepare'd'according to this reaction byemploying the appropriate Schitfs base are N -benzoyl-N -formylpteroylglutamic acid, N -butyryl-N formyl pterolyglutamic acid, N-propionyl-N formyl pteroylglutamic acid and N -phenylacetyl-N -formylpteroylgutamic acid.

According to the next and last step, pteroylglutamic acid is produced byhydrolyzing N -acyl-N -formyl pteroylglutamic acid with an inorganic ororganic acid or base. It is preferred however, to employ the commoninorganic acids and bases such as sodium hydroxide, potassium hydroxide,hydrochloric acid, sulfuric acid, and phosphoric acid. The reaction isrun by intimately contacting the reactants in a suitable solvent such aswater or an inert organic solvent. Elevated temperatures can be used, ifdesired, to promote the reaction. After the hydrolysis has beencompleted the pteroylglutamic acid is isolated from the reaction mixtureby conventional methods.

Alternatively the N -acyl-N -formyl pteroylglutamic acid (X) can beproduced by reduction of N-{lp-f (2-acylamino-4-hydroxy-o-pteridylmethylene) imino] benzoyl} glutamic acid(IX) with triethyl ammonium formate. The reaction is effected byintimately contacting the reactants at an elevated temperature,preferably at about C. Although a solvent may be added it is generallypreferred to use an excess of triethyl ammonium formate as the reactionmedium. After the reaction has gone to completion the product isrecovered by conventional methods such as adding water to precipitate,the N -acyl-N -formyl pteroylglutamic acid and filtering. By followingthis procedure a large number of li -acyl N- -formyl pteroylglutamicacids can he, prepared in which the acyl group is an alkyl, aryl, oraralkyl carbonyl radical. Examples of compounds which can be prepared inthis fashion by using the appropriate Schiifs base are N -acetyl-N-formyl pteroylglutarnic acid, N -benzoyl- N -formy1 pteroylglutamicacid, N. -butyryl-N -formy1 pteroylglutamic acid, and N -phenylacetyl-N-formyl pteroylglutamic acid.

Pursuant to an additional embodiment of the invention it has been foundthat N -acyl-N -formyl pteroylglutamic acid (X) can be produced byintimately contacting 2,- acy1amino-4-hydroxy-.6.-forn y1 pteridine.(VII) and N te-am no z a mam as wi l) in t e presence or formic acid.This reaction can be illustrated as follows:

wherein R? i an alkyl carbonyl, aryl carbonyl, or afalkyl carbonylradical. The reaction is effecte in a suitable reaction medium which canbe furnished by an added inert organic solvent or use of an excess 1 offormic acid.- Examples of solvents which can be used are dioxane and thelower alcohols such as ethanol and propanol; An elevated temperature,such as the reflux temperature, is prefcrably used to aid dissolution ofthe reactants and complete the reaction in a short period of time. In aspecific embodiment of this process N -acetyl-N -formyl pteroylglutamicacid is produced by condensing 2-acetamido-4- hydroxy-6-iormylpteridine, L(-l)N-(p-arninobenzoyl) glutamic acid and formic acid indioxane. In another specific application of this process N -acetyl-N-formyl pteroylglutamic acid is formed by reacting Z-acetamido-4-hydrcxy-6-formyl pteridine with L(-l)N-(p-amiuoben zoyl) glutamic acidin an excess of formic acid. Illustrative of other N -acyl-N -formylpteroylglutamic acids which can be produced in this way are N-propionyl-N forrnyl pteroylglutamic acid, N -benzoyl-N -formylpteroylglutamic acid, and N -phenylacetyl-N -formyl pteroylglutamicacid.

According to a further embodiment of this invention N -acyl-N -formylpteroylglutamic acid (X) is produced by reacting2-acylamino-4-hydroxy-6-formyl pteridine (VII) withL(-'l)N-(p-formamiriobenzoyl) glutamic acid (XII). This reaction may beillustrated as follows:

CHO

oobn XII wherein R is anacyl group such as an alkyl carbonyl, arylcarbonyl, or aralkyl carbonyl radical. Examples of substitutedpteridines which can be used in this reaction areZ-acetamido-4-hydroxy-6-formyl pteridine,2-phenylacetamido-4-hydroxy-6-formyl pteridine, 2-benzamido-4-hydroxy-6-formyl pteridine, and 2-butyramido-4-hydroxy- 6-formylpteridine; Formation of the N -acyl-N -formyl pteroylglutamic acid isaccomplished by intimately contacti'ng the reactants ina suitable liquidreaction medium at an elevated temperature. Some suitable solvents inwhich to conduct the reaction are dioxane, the lower alcohols, water,and formic acid. In general, formic acid of approximately 98% purity hasgiven the best results. Elevated temperatures of 60l00 C. have beenfound satisfactory although higher and'lower temperatures may be used inspecial circumstances. In a specific embodiment of this reaction N-acetyl-N -forrnyl pteroylglutamic acid is prepared by condensing2-acetamido-4-hy-' droxy-G-torrnyl pteridine withL(-1)N-(p-formamidobenzoyl) glutamic acid in 98% formic acid at about C.The L(-l)N-(p-formamidobenzoyl) glutamic acid may be readily prepared byheating L(-1)N-(p-aminobenzoyl) glutamic acid with concentrated formicacid at a temperature of about 60l00 C.

It has further been found that a N -acyl-N -formyl pteroylglutamic acid(X) can be prepared by reacting a 2- acylamino pteridine having in the 4position a hydroxy, alkoxy, aryloxy or aralkoxy radical and in the 6position a dialkoxymethyl, diaryloxymethyl, or diaralkoxymethyl radical(VI) with L(-l}N-(p-aminobenzoyl) glutamic acid (VIII) andformic acid ora source of formic acid such as triethyl ammonium formate. Under suchconditions the 6-acetal is converted in situ to a 6-formyl group. Thisreaction may be illustrated as follows:

cum Goon L HzN-Q-C o b11185 WEN N/ c oim v1 vrn PJHN-k 0 0 0 II or anadded inert organic solvent, examples of which are. dioxane, the loweralcohols, methyl Cellosolve and the saturated hydrocarbons such ashexane. Elevated temperatures are used to promote the reaction, therange 70-100 C. having been found quite satisfactory. After the reactionis completed the product is isolated by ordinary procedures. In aspecific application of this method N -acetyl-N -formyl pteroylglutamicacid is formed by reacting 2-acetamido-4-hydroxy-6-diethoxymethylpteridine with L(-l)N-(p-aminobenzoyl) glutarnic acid in 98% formic acidat about 70 C. Examples of other starting materials which can be reactedwith L(-l)N- (p-aminobenzoyl) glutamic acid to produce the correspondingN -acyl-N -formyl pteroylglutamic acid are 2-acetamido-4-benzyloxy'6-dipropoxymethyl pteridine, 2-

benzamido-4-phenoxy-6-dibenzyloxymethyl pteridine, and 2 phenylacetamido4 propoxy 6 dimethoxy methyl pteridine.

In yet another embodiment of the invention N -formyl pteroylglutamicacid (XIII) can be produced by reacting a non-acylated 2-amino pteridinehaving in the 4 positions a hydroxy, alkoxy, aryloxy, or arallcoxyradical and in the 6 position a dialkoxymethyl, diaryloxymethyl, ordiaralkoxymethyl radical (V) with L(-l )ll-(p-aminobenzoyl) glutamicacid (VIII) in an excess of formic acid. This reaction is illustrated asfollows:

/N 00011 CH2N- CONHCH 1 I CH2 The reaction is wherein R is an alkoxy,aryloxy, or aralkoxy radical, and hydrogenation of the correspondingSchitfs base, N-{p R is a hydroxy, alkoXy; armor or aralkoxy radical.Some specific examples of the substituted Z-amino pteri= dines which canbe used as starting materials are Z-amino- 4-methoxy-6-diethoxymcthylpteridine, 2-amino-4-phebe illustrated as follows:

noXy-6-dipropoxymethyl pteridine, 2-amino=4-benzy10xy- R Y 6dib'enzyloxy pteridine, and 2-amino-'4-ethoxy-6-di- 8 methoxy'methylpteridine. Thereaction is conducted'in T an excess of substantially pureformic acid at an elevated R2HN L SE temperature to promote thereaction, a satisfactory ter'n- 10 \N/\N// peratuie being 70-80 c. It ispreferred to employ 98% IX formic acid containing a small amountofacetic anhydride R to obtain the highest yields. In a specificapplication of A N coo-H this process N -formyl pteroylglutamic acidisfforr'ned N I 0HNHT GONHCE, by reacting2-amino-4-hydroxy-6-diethoxyrnethy1 pterik i on,- dine withL(-1)N-(p-aminobenzoyl} gliitan'iic acid in an yy 000E excess of formicacid at about 70 C.

Ii i stillanother embodiment of the invention N -forrnylpteioylghi't'ainic acid and N -acyl derivatives thereof (X) wherein R2represents y p- The usual y a'n b p r d b e in a zami o1- 2- 1 in .'4genation catalysts may be used to effect the reaction alhydi'dxy'.fiformyl pteiidine v11 wi -1)N.( though'it is preferred to use Raneynickel. The hydroaminobenzoyl) glutamic acid (VIII) in the presence ofgenation is conducted in a suitable reaction medium such trieth'ylammonium format'e as .a source of formic acid. s wa dioxane, glacialacetic acid, quinoline, a lower This reaction can be illustrated asfollows:

XIV

XIII, or X when R is acyl CH COOH wherein R represents a hydrogen oracyl radical; The generally employed to aid the reaction. According tostarting materials which may be used in this'reactiOn are one embodimentof this process N -acetyl pteroylglutamic 2 amino-4-hydroxy-6-formylpteridine and derivatives acid is produced by hydrogenatingN-{pKZ-acctamidothereof having an acyl radical in the 2 position whichcan 4-hydroxy-G-pteridylmethylene) imino] benzoyl}glutamic be either analkyl carbonyl, aryl carbonyl; or aralkyl carboh'yl group. Examples ofsuitable starting materials in this class are Z-piopiohamido-4-hydroXy-6formyl pteridine, 2-benzamido-4-hydroxy 6-formyl ipt'eridi'ne, 2-benzoyl}glutamic acid, N-{p-I(2-benzamido-4-hydroxy-6-buty'ramido-4-hydroXy-6 formyl pte'ridin'e, and 'Z-phenyL p i y y iminoly glutamic acid, and acetam'idb-4-hydroxy-6-formyl pteridine. Thedesired N :p butyramido 4 Y Y 6 Pteridyl- N formyl pteroylglutamic acidor its N -acyl derivatives methylene) iminol henZ'OYUghItamic acid amalso are formed by intimately contacting the reactants at an duccd tothe corresponding N -acyl pteroylglutamic acid. elevated temperature,for example 150 C. or above. The resulting N -acyl pteroylglutarnic acidmay be readily Temperatures substantially below 150 C. are notprehydrolyzed by an inorganic 01' organic acid or base to ferred toeffect the reaction. Atmospheric pressure or ptefroylglutamic aci'd.super atmospheric pressures can be employed. After the It has furtherbeen found that p-[N-(2-amino--- reaction is completed the reactionmixture can be worked hydroxy 6-pteridylmethyl) forrriarnido] benzoicacid, up according to conventional methods to obtain the prodcommonlycalled rhizopterin, and N -acyl derivatives uct. In a specificapplication of this reaction N' -forrnyl thereof (XV) can be readilyproduced by reacting a 2- pteroylglutamic acid is formed byreactingZ-acetamido-4- amino or 2-a'cyl'amino pteridine having in the4-position a h'ydrox'y-6-formyl pteridine with L1)'N-(p-ami1iobenhydroxy alkoxy, aryloxy or aralkoxy radical and in thezoyl)' -glutamic acid in triethyl ammonium for-mate at 6:.positionadi'alkoxymethyl, diaryloxymethyl, or diaral- C. v I 5 koityme'thylradical (V or VI) with p-aminobenzoic acid In one other embodiment ofthis invention N -acy1 and an excess of formic acid. This reaction maybe illuspteroylglutamic acid (XIV) can be produced by catalytic tratedas follows:

this process other Schiifs bases such as N-{p-[(2-phenylacylamino 4hydroxy 6 pteridylmethyleney iminolbenzoyl'} glutamic' acid (IX). Thisreaction may alcohol, and the like; Super atmospheric pressures areacid, with Raney nickel in water. By the procedure ofacetarnido 4hydroxy 6 pteridylmethylene) imino] 11 wherein. R represents an alkoxy,aryloxy, or aralkoxy radical, R represents a hydroxy, alkoxy, aryloxy,or aralkoxy radical, and R represents hydrogen or an acyl radical.Examples of pteridines within this class which are useful startingmaterials are 2-amino-4-methoxy-6- diethoxymethyl pteridine,2-acetamido-4-phenoxy-6-dipropoxymethyl pteridine,2-amino-4-benzyloxy-6-dibenzyloxymethyl pteridine, andZ-phenylacetamido-4-ethoxy-6- dimethoxymethyl pteridine. Formation ofthe desired p- [N-(2-amino-4-hydroxy-6pteridylmethyl) formamido] benzoicacid or N -acyl derivatives is conveniently achieved by intimatelycontacting the reactants at an elevated temperature. A temperature of60100 C. has been found suitable for this purpose at atmosphericpressure. The product may be recovered from the reaction mixture byconventional methods such as adding ether to precipitate the product.

The examples which follow illustrate methods of carrying out the presentinvention but it is understood, however, that these examples are not tobe considered as limiting the invention.

This application is a continuation-in-part of Serial No. 334,382, filedIanhary 30, 1953, now abandoned.

EXAMPLE 1 Production of 2-br0mo-3,3-diethoxy-propi0naldehyde 26 gm. of2-bromo-3,3-diethoxy propionaldehyde having I a boiling point of 63-65C. at 2.5 mmpof mercury; n -=1.4513.

The l.2-dibromo 1,3,3-triethoxy propane used as the starting materialwas prepared by reacting 50 gm. of l,3,3-triethoxypropylene-1 in 125 ml.of ether with 41 gm. of bro-mine while stirring and maintaining the temperature between -10 C. The solution was stirred for 1 hour at 0-10 C.and then the solvent removed under reduced pressure to produce a residueof 1,2-dibromo- 1,3,3-triethoxy propane.

EXAMPLE 2 Production of Z-chl0ro-3,3-diethoxy-propionaldehyde To a 100ml. of 3-necked flask equipped with stirrer, thermometer, and gas inletwas added 34.8 gm. of 1,3,3- tri-ethoxy-propylene-l. It was cooled withstirring to about 5 C. and then 13.6 gm. of chlorine was bubbled throughthe liquid over a period of 45 minutes. The reaction mixture containing1,2-dichloro-1,3,3-triethoxy propane was then added under nitrogen andwith stirring to a mixture of 21 ml. of water, 62.5 ml. of dioxane, and26.4 gm. 'of sodium bicarbonate. The reaction mixture was maintained at5-10" C. and stirred for 2 hours. The reaction mixture was extractedtwice with 50 ml. portions of ether. The combined ether extracts werewashed with water and dried over sodium sulfate. The ether solution wasfiltered and removed under reduced pressure. The residue was distilledover calcium carbonate under reduced pressure to give2-chloro-3,3-diethoxy-propionaldehyde having a boiling point of 58-60"C. at 1.2 mm. of mercury; H '=1.4309.

In the same manner, 2-chloro-3,3-dipropoxy-propionaldehyde,2-chloro-3,3-dibenzyloxy-propionaldehyde and2-chloro-3,3-dibutoxy-propionaldehyde can be prepared by employing thecorresponding 1,2-dichloro substituted propane.

acid added to dissolve most of the oil.

12 EXAMPLE 3 Production of 2-amin0-4-benzyloxy-6-diethoxymethyl-5,6-dihydr0pteridine To the resulting slurry 2.5 N hydrochloric acid was7 added until the mixture became acidic. Insoluble matter that formedwas removed by filtration. After cooling, the filtrate was added toanexcess of 6 N ammonium hydroxide at a temperature of 5-l0 C. Theamorphous precipitate was removed by filtration and dried. The yield ofZ-amino-4-benzyloxy-6-diethoxymethyl-5,6-di.hydropteridine was 3.5 gm.

A sample was purified further by dissolving it in ethyl acetate andadding n-hexane until the product precipitated.

EXAMPLE 4 Production of 2-aminc-4-benzyloxy-6-dieth0xymethyl pteridineTo an ethanol solution of2-amino-4-benzyloxy-6-diethoxymethyl-S,G-dihydropteridine was added mg.of ferrous sulfate in 1 ml. of water and then 2.3 gm. of 30% hydrogenperoxide in 10 ml. of water was added over a 30 minute period. Themixture was concentrated under reduced pressure to a small volume and2.5 N hydrochloric The solution was separated from insoluble material bydecantation and added to an excess of cold 6 N ammonium hydroxide.

i hydroxy-6-diethoxymethyl-S,6-dihydropteridine.

A precipitate resulted which was isolated and dried. The yield of2-amino-4-benzyloxy-G-diethoxymethyl pteridine was 4 gm. and itsultraviolet absorption spectrum in 0.1

N NaOH had maxima at 2560 A. (E%=369) and 3610 A. (E% =2l0); in 0.1 NHCl it had a maximum at 3350 A. (E% =364).

By a paper strip chromatography, run according to the procedure ofWeygand in Experientia 6, 184 (1950), of the acid obtained bypermanganate oxidation of 2- amino-4-hydroxy-6-formyl pteridine whichwas produced by metal hydrolysis of 2-amino-4-benzyloxy-6-diethoxymethylpteridine, it was found that the product was all the desired position 6isomer.

The solution of 2 amino 4 benzyloxy 6diethoxymethyl-5,6-dihydropteridine used in this example was prepared byreacting 4.6 gm. of 2,4,5-t1iamino-6-benzyloxy pyrimidine and 1.8 gm. ofsodium acetate in 63 ml. of ethanol with 4.5 gm. of2-bromo-3,3-diethoxy-propionaldehyde in 63 ml. of ethanol.

EXAMPLE 5 Production of 2-amino-4-hydroxy-6-diethoxymethyl-5,6-dihydroptridine and 2 amino 4 hydroxy 6 diethoxymethyl pteridine 5.0 gm.of 2,4,5-triamino-6-hydroxy pyrimidine sulfate was dissolved in ml. ofwater containing 5 gm. of barium chloride. The solution was heated to 60C. in

a nitrogen atmosphere for 1 hour with stirring and subsequently filteredhot to remove the insoluble precipitate.

To the resulting solution of 2,4,5-triamino-6-hydroxy pyrimidine wasadded 140 ml. of ethanol and then 5.14 gm. of2-bromo-3,3-diethoxy-propionaldehyde. The reaction mixture was stirredunder nitrogen at room temperature for 56 hours to yield a solution of2-amino-4- I The solution was then adjusted to pH 8-9 and 50 mg. offerrous chloride and 2.6 gm. of 30% hydrogen peroxide added at roomtemperature. The solution was stirred 5 hours,

13 filtered, and the precipitate washed with water, alcohol, and etherto'yield purified 2-amino 4-hydroxy-6-diethoxymethyl pteridine.

A sample was purified by conversion to the sodium salt andreprecipitation of the original free base. It had an ultravioletabsorption curve which exhibited maxima in 0.1. N HCl at 3180 A. (E%=340) and in 0.1 N NaOH at 2550 A. (E%=941), 3620 A. (E%=288).

In a similar manner, 2-amino-4-hydroxy-6-diethoxymethyl. pteridine wasprepared by reacting 2-chloro-3,3- diethoxy-propionaldehyde with2,4,5-triamino-6-hydroxy pyrimidine in ethanol and in the presence ofsodium acetate to produce 2-amino-4-hydroxy-6-diethoxymethyl-5,6-dihydropteridine which was oxidized with hydrogen peroxide andferrous sulfate to 2-amino-4-hydroxy-6-diethoxymethyl pteridine.

EXAMPLE 6 Production of Z-umino-4-hydr0xy-6-dibenzyloxymethyl- 5,6dihydropteridine and 2 amino 4 hydroxy 6- dibenzyloxymethyl pteridineAfter passing nitrogen through a stirred solution of 8.2 gm. of sodiumacetate in a mixture of- 200ml. of ethanol for one-half hour, 4.7 gm. of2,4,5-triamino-6-hydroxy pyrimidine dihydrochloride and 7.0 gm. of2-bromo-3,3-

EXAMPLE 7 Production of 2-acetamido-4-hydroxy-6-diethoxymethyl pteridineTo a 3-necked flask equipped with stirrer and reflux condenser was added4.2 gm. of 2-amino-4-hydroxy-6-diethoxymethyl pteridine and 80 ml. ofacetic anhydride. The mixture was refluxedwith stirring for 1.5 hours.To the solution was added 2 gm. of activated carbon and refluxing wascontinued for 10 minutes. The hot solution was filtered and cooled inthe icebox overnight. The crystalline precipitate which formed wasfiltered and washed with 10 ml. of cold acetic anhydride and then withether. The precipitate was dried at 50 C. under reduced pressure toyield white crystalline 2-acetamido-4- hydroxy-6-diethoxymethylpteridine. A 1 gm. sample of the product was recrystallized from 2 ml.ofdioxane and melted at 198200 C.

The ultraviolet absorption curve exhibited maxima in 0.1, N NaOH at 2560A. (E%=850) and 3500 A. (E% :250).

EXAMPLE 8 Production of 2-propionamido-4-hydroxy-6-dieth oxymethylpteridine A slurry of 3.5 gm of 2-amino-4-hydroxy-6-diethoxymethylpteridine in 70 gm. of propionic anhydride was heated at 140 C. for fivehours during which time solution was elfected. To the reaction mixturewas added 1.5 gm. of charcoal and the mixture filtered. The filtrate wasevaporated to about one-half volume and allowed to stand overnight inthe cold. The solid was collected by filtration, washed twice with ethylether and twice with petroleum ether. After drying in air the light tancrystals of 2-propionamido-4-hydroxy-6-cliethoxymethyl pteridine weighed295 gm.

14 EXAMPLE 9 Production of 2-acetamido-4-hydroxy-6-formyl pteridine Toaflask equipped with a stirrer Was. added 860 ml'. of 98% formic acidand 58 gm. of 2-acetamido-4-hydroxy-6-diethoxyrnethyl pteridine.Complete solution was achieved in 5 minutes. Upon standing for 15minutes at room temperature a precipitate appeared. The solution wasallowed to stand under nitrogen in an ice-box overnight. It was filteredand the precipitate was washed with cold formic acid and thenanhydrous'ether giving a formic acid salt of2-acetamido-4-hydroxy-6-formyl pteridine.

The ultraviolet absorption curve exhibited maxima in- 0 .1 N NaOH at2550 (E%=809) and 3500 A. (E%=294). The salt loses formic acid whenheated at 100 C. for three hours at 3 mm. pressure.

EXAMPLE 10 Production ofN-{p-[(2-acetamido-4-hydroxy-6-pteria'ylmethylene) imino]-benzoyl}glutamic acid To a 1 liter flask equipped with stirrer and refluxcondenser containing 660 ml. of ethanol was added 6.6 gm. of L(-l)N-(p-aminobenzoyl)- glutamic acid. The mix ture 'was refluxed in a nitrogenatmosphere until solution was complete (20' minutes)". At this point 5.3gm. of Z-acetamido-4-hydroxy-6-formyl pteridine was added. Refluxing andstirring were continued for 1 hour during which th e aldehyde dissolved;The solution was filtered while hot. and allowed to stand overnight atroom temperature. A yellow crystalline precipitate formed which wasfiltered and dried under reduced pressure at 100 to remove adsorbedsolvent to produce N-{p-[(2-acetamido-4-hydroxy-6-pteridylmethylene)imino} benzoyl} glutamic acid.

The ultraviolet absorption curve exhibited maxima in- 0.1 N NaOH at2580A. (E%-=627), inflection 2750 A. (E%=505), and 3500 A. (E%=156).

EXAMPLE 11 Production of N -acetyl-N -formyl pteroylglutumic acidEXAMPLE 12 Production of N -acetyl-N -formyl pteroylglutamic acid byreduction of N-{p-[(-2acctamido-4-hydr0xy-6 pteridylmethylene) imino]benzoyl} glutamic acid with triethyl ammonium formate A mixture of 0.96gm. of N-{p-[(2-acetamido-4-hy droxy-6-pteridylmethylene) imino]benzoyl} glutamic acidin 7.0 gm. of triethyl ammonium formate was heatedat C. for two hours. After standing overnight at roomtemperature, thereaction mixture was diluted with 100 ml. of water and cooled in therefrigerator for several hours. The N -acetyl-N- -formyl pteroylglutamic acid was recoveredby filtration and washed with water, acetone,and ether. The product was assayedwithL. casei and found to possessfolic acid activity.

The resulting yellow solution was heated I EXAMPLE 13 Production of N-acetyl-N -formyl pteroylglutamic acid by condensing2-acetamido-4-hydroxy-6-formyl pteridine withL(-1)N-(p-aminobenzoyl)glutamic acid in dioxane in the presence offormic acid To a boiling solution of 60 ml. of dioxane was added 600 mg.of L(-1)N-(p-aminobenzoyl) glutamic acid and 400 mg. of2-acetamido-4hydroxy-6-formyl pterid ine and the mixture refluxed untilsolution was complete. To the mixture was added a solution of 18 ml. offormic acid and 2 ml. of acetic anhydride. The reaction mixture wasrefluxed for one hour, cooled, and 300 ml. of ether added to precipitatethe N -acetyl-N -formyl peteroylglutamic acid. It was filtered, washedwith ether and dried. The product assayed 59% folic acid against L.casci.

EXAMPLE 14 Production of N -acetyl-N -formyl pteroylglutamic acid bycondensing 2-acetamido-4-hydroxy-6-formyl pteridine withL(-1)N-(p-formamidobenzoyl) glutamic acid ,To a solution of 400 mg. of2-acetamido-4-hydroxy-6- iormylpteridine in 18 ml. of 98% formic acidcontaining a small amount of acetic anhydride was added 600 mg. ofL(-1)N-(p-formamidobenzoyl) glutamic acid. The mixture was heated at65-70 C. for one hour and then cooled. To this solution was added 200ml. of ether to precipitation N -acetyl-N -formyl pteroylglutamic acid.The solid was filtered, washed with ether and dried. The productpossessed folic acid activity to L.

onset.

The L(-l)N-(p-formamidobenzoyl) glutamic acid used in this reaction wasproduced by heating gm. of L(-l )lfl- (p-aminobenzoyl) glutamic acid in25 ml. of 85% form1c acid for one hour. On cooling a white precipitateof L(-1)N-(p-formamidobenzoyl) glutamic acid separated which wasfiltered, washed with ether and dried. It melted at 190 C.

EXAMPLE 15 EXAMPLE 16 Production 0 N -formyl pteroylglutamic acid To 35ml. of formic acid was added 1.86 gm. of 2-amino-4-benzyloxy-6-diethoxymethyl pteridine, and 1.4 gm. ofL(-1)N-(p-aminobenzoyl) glutamic acid. The reactants dissolved readilyto give a dark red solution which was heated at 67 C. for one hour. Thereaction mixture was cooled to 20 C. and poured into 400 ml. of etherwith stirring. The resulting precipitate was washed with formic acid,ether, water, and ether. The product 0btaiued contained N -formylpteroylglutamic acid and had 23% folic acid activity when assayed withS. faecalis.

EXAMPLE 17 Production of N -formyl pteroylglutamic acid by reacting-2-amino-4-hydroxy-6-formyl pteridine with L(-1)N-(paminobenzoyl)glutamic acid in triethyl ammonium formate A mixture of 1.0 gm. of2-amino-4-hydroxy-6-formyl pteridine, 1.4 gm. of L(-1)N-(p-aminobenzoyl)glutamic acid, and 15 gm. of triethyl ammonium formate was heated at 150C. for one hour. The reaction mixture was concentrated under reducedpressure below C. and diluted with 100 m1. of water. The N -formylpte'roylglutamic acid precipitated from the reaction mixture and wasrecovered by filtration. It was washed with water, acetone, and etherand dried in air. The product has folic acid activity when assayed withL. casei and S. faecalis.

EXAMPLE 18 Production of N -acetyl-N -formyl pteroylglutamic acid byreacting Z-acetamido-4-hydr0xy-6-formyl pteridine withL(-1)N(p-aminobenzoyl) glutamic acid in triethyl ammonium formate To asolution of 466 mg. of 2-acetamido-4-hydroxy-6- formyl pteridine in 13m1. of triethyl ammonium formate was added 600 mg. ofL(-1)N-(p-aminobenzoyl) glutamic acid. The mixture was heated to 150 C.for one hour and then concentrated under reduced pressure. About 100 ml.of acetone was added to the concentrate and a precipitate formed. The N-formyl pteroylglutamic acid was recovered by filtration and washed withacetone and ether and dried. It was found to have folic acid activitywhen assayed with L. casei.

EXAMPLE 19 Production of N -acetyl pteroylglutamic acid by catalyticreduction of N-{p-[ (2-acetamido-4-hydroxy-6-pteridylmethylene) imino]benzoyl} glutamic acid EXAMPLE 20 Production ofp-[N-(Z-amin0-4-hydr0xy-6-pteridylmethyl) formamido] benzoic acid To ml.of formic acid was added 3.5 gm. of 2-amino-4-benzyloxy-6-diethoxymethyl pteridine and 1.4 gm. of p-aminobenzoicacid. The mixture was heated at 67 C. for 1 hour, cooled, and added to 1liter of ether with stirring. The reaction mixture was centrifuged andthe precipitate recovered. It was washed with alcohol, ether and thendried to produce p-[N-(2-amino-4-hydroxy-6- pteridylmethyl) formamido]benzoic acid.

1 gm. of p-[N-Z-amino-4-hydroxy-6-pteridylmethyl) formamido] benzoicacid and 5 gm. of sodium bicarbonate were added to 35 ml. of water. Themixture was heated on a steam bath with shaking for 10 minutes. Thereaction mixture was filtered hot. To the filtrate, acetic acid wasadded until the solution was acidic. The mixture was centrifuged and thesolid washed and dried to produce purifiedp-[N-(2-amino-4-hydroxy-6-pteridylmethyl) formamido] benzoic acid as ayellowish powder.

The product had an ultraviolet absorption spectrum in 0.1 N NaOI-I withmaxima at 2560 A. (E% =479) and 3630 A. (E%=200) and in 0.1 N HCl at2480 A. (E%=3l1) and 3170 A. (E%=205).

The product had a microbioligical activity of SOO'y/mg. against S.faecalis.

EXAMPLE 21 Hydrolysis of Z-acetamido-4-hydr0xy-6-diethoxy methylpterzdme to 2-acetamido-4-hydroxy-6-f0rmyl pteridine in 2.5 N HCl.

5 grams of 2-acetamido-4-hydroxy-6-diethoxymethyl 17 pteridine was addedall at once to a stirred solution of 100 ml. of 2.5/N HCl. Solun'on ofthe solid occurred and then a precipitate of2-ac-etarnido-4-hydroxy-6-formyl pteridine deposited. This precipitatewas filtered and washed with water, acetone and ether.

EXAMPLE 22 Production of N-{p-[(2-acetamido-4-hydr0xy-6-pteridylmethylene) imi'no] benzoyl} glutamic acid-condensation in dioxane 1.8grams of p-aminobenzoyl glutamic acid and 180 ml. of dioxane wererefluxed until all solid dissolved.

To this solution while hot was added 1.4 grams of2-acetamido-4-hydroxy-6-formyl pteridine. n refluxing for 25 minutespractically all the solid dissolved. The solution was filtered from someinsolubles. On standing at room temperature a yellow precipitate of N {p[(2 acetamido 4-hydroxy-6-pteridylmethylene) imino] benzoyl} glutamicacid came down. This was filtered and dried at 100 C. /2 mm.

Various changes and modifications of the invention can be made and, tothe extent that such variations incorporate the spirit of thisinvention, they are intended to be included within the scope of theappended claims.

What is claimed is:

1. A 2-amino-4-R'-6-di R methyl pteridine, wherein R represents a memberselected from the class consisting of lower alkoxy and benzyloxyradicals and R represents a member selected from the class consisting ofhydroxy and benzyloxy radicals.

2. A 2-amino-4-benzyloxy-6-di-lower pteridine.

3. 2-amino-4-bienzyloxy-6-diethoxymethy1 pteridine.

4. A 2-amino-4-hydroxy-6-di-lower alkoxymethyl pteridine.

5. 2-amino-4-hydroxy-G-diethoxymethyl pteridine.

6. Z-amino-4-hydroxy-6-dibenzyloxymethyl pteridine.

alkoxymethyl References Cited in the file of this patent UNITED STATESPATENTS 2,500,296 Waller et a1 Mar. 14, 1950 2,520,156 Lindlar et a1Aug. 29, 1950 20 2,599,526 Cosulich June 10, 1952 OTHER REFERENCESRichter: Textbook of Organic Chemistry, 1st edition (1938) pp. 103-4.

1. A 2-AMINO-4-R''-6-DI R METHYL PTERIDINE, WHEREIN R REPRESENTS AMEMBER SELECTED FROM THE CLASS CONSISTING OF LOWER ALKOXY AND BENZYLOXYRADICALS AND R'' REPRESENTS A MEMBER SELECTED FROM THE CLASS CONSISTINGOF HYDROXY AND BENZYLOXY RADICALS.